Influenza virus continues to be a major threat to global human health. The persistent episodes of direct transmission of highly pathogenic avian influenza strains to humans, the high prevalence of resistant strains to current anti-influenza drugs among H1N1 human strains, and the high genetic compatibility between swine- origin H1N1 and highly pathogenic avian H5N1 strains, suggest that the odds of humanity facing the emergence of a deadly drug-resistant pandemic strain remain high. A plausible new strategy to develop broad- spectrum anti-influenza therapies is to target cellular components required by the virus. In our previous studies, we evaluated the relevance of the cellular SUMOylation system for influenza infection. Our data identified several influenza proteins as bona fide SUMO targets, revealed the ability of the virus to trigger a two-fold global increase in cellular SUMOylation, and demonstrated that large global changes in cellular SUMOylation affect viral protein expression and virus multiplication. Therefore, the cellular SUMOylation system appears to play an important role for influenza virus infections. Importantly, out of all viral SUMO targets identified, the non-structural protein NS1 was the most efficiently SUMOylated. Considering this fact, the numerous roles played by NS1 during viral infection, and the functions normally associated to SUMOylation, we hypothesize that SUMOylation regulates NS1 function by affecting its ability to establish specific protein-protein interactions with other viral and cellular proteins, thus explaining its relevance for influenza infection. To test this hypothesis, we will evaluate the effects exerted by NS1 SUMOylation on two main parameters: i) NS1's ability to interact with other viral and cellular proteins. ii) The main functional and biochemical characteristics of NS1, namely its ability to neutralize type-I interferon responses, its cellular localization, and its half-life. These studies will be pursued using a combination of protein mutants, viral mutants, stably transduced cell lines, and an artificial modulator of NS1 SUMOylation. The data generated will provide critical insights on the post- translational regulation of NS1 function and the molecular effects mediated by SUMOylation during influenza virus infection, and potentially lead to the identification of novel targets for the development of broad-spectrum anti-influenza therapies. Additionally, these studies will also expand our understanding of the interactions established between viruses and the cellular SUMOylation system. PUBLIC HEALTH RELEVANCE: Influenza remains a major infectious disease responsible for annual epidemics and capable of producing extremely damaging pandemics. A better knowledge of the multiple interactions established between the virus and its host cell will likely lead the way to the development of more effective treatments. This study will characterize at the molecular level the effects exerted by a host's post-translational modification system upon the non-structural protein NS1, potentially leading to the identification of novel antiviral targets.